A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans

A61K38/22—Hormones

A61K38/26—Glucagons

A—HUMAN NECESSITIES

A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE

A61K—PREPARATIONS FOR MEDICAL, DENTAL, OR TOILET PURPOSES

A61K9/00—Medicinal preparations characterised by special physical form

A61K9/0012—Galenical forms characterised by the site of application

A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner

A61K9/0024—Solid, semi-solid or solidifying implants, which are implanted or injected in body tissue

Abstract

A pharmaceutical preparation comprising a suspension of glucagon in the presence of a metallic cation and optionally an excipient provides for a sustained release of glucagon into the circulation of a treated patient following injection of pharmaceutical preparation.

Description

PATENT APPLICATION

EXTENDED RELEASE FORMULATIONS OF GLUCAGON AND OTHER

PEPTIDES AND PROTEINS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. provisional application No. 60/900,509, filed February 8, 2007, the entire content of which is incorporated herein by reference.

FIELD OF THE INVENTION

[0002] The invention relates to extended release pharmaceutical formulations for glucagon and other peptides and proteins. The invention relates to the fields of medicine and pharmacology.

BACKGROUND OF THE INVENTION

[0003] Glucagon is a polypeptide hormone secreted by alfa-cells of the pancreatic islets of Langerhans. Human glucagon is a single-chain polypeptide consisting of 29 amino acid residues, the sequence of which is published, inter alia, in The Merck Index, 10th Edition (1983), Monograph No. 4307, incorporated herein by reference.

[0004] Glucagon is used for the treatment of hypoglycemia in diabetics due to its glycogenolytic effect on the liver. Glucagon also exerts a spasmolytic effect on smooth muscles, which is used clinically in connection with several imaging procedures, especially radiology.

[0005] Glucagon is at present marketed in the form of a lyophilized product for injection comprising lactose as the sole excipient. The lyophilizate is reconstituted using a suitable diluent.

[0006] When used for the treatment of hypoglycemia, the currently marketed form of glucagon has a plasma half life of approximately 17 minutes. Recently, however, a new use of glucagon for the prevention of hypoglycemia, particularly for those at risk
of suffering insulin-induced hypoglycemia as a result of diabetes therapy, has been described (see U.S. Patent No. 7,314,859, PCT Pub. Nos. WO 06/004696 and WO 04/060387 and U.S. Pat. App. Pub. No. 20060014670, each of which is incorporated herein by reference). For the prevention of hypoglycemia, glucagon levels are maintained at a basal level over extended periods of time to provide a buffering effect with respect to the action of insulin. As described in the above patent publications, the prevention of hypoglycemia with glucagon can be achieved with a basal level of glucagon, which is much lower than the relatively high levels that result from administering the currently marketed glucagon as directed for the prevention of hypoglycemia. Currently marketed glucagons provide for only an immediate, short- acting release of relatively high doses glucagon and so are not ideally suited for use in the prevention of hypoglycemia.

[0007] Excessive glucagon can increase plasma glucose, which is a cause of the secondary complications of diabetes. These secondary complications of diabetes include microvascular diseases such as retinopathy and nephropathy and microvascular diseases such as cardiovascular disease. Thus, a pharmaceutical preparation appropriate for the prevention of hypoglycemia must be able to provide for prolonged exposure to glucagon without inducing an excessive increase in plasma glucagon (and therefore, glucose) levels.

[0008] Production of conventional pharmaceutical preparations of glucagon requires utmost care to avoid undesired decomposition of glucagon during preparation, allocation into drug product containers, and lyophilization. Currently marketed glucagon undergoes decomposition during storage of the finished product at room temperature, significantly limiting the shelf-time of any ready-for-use preparation. Consequently, most marketed glucagon is sold in a lyophilized form and must be reconstituted before use.

[0009] The scientific literature has described formulations of glucagon with "prolonged action" that contain either zinc or zinc/protamine (see, e.g., Trading et al., 1969, EMr. J. Pharm. 7:206-210); however, these formulations have not been approved by any regulatory authority for human use. Further, as plasma glucagon was not determined after administration of these glucagon formulations to dogs or to
humans, it was not shown that there was an extended release of glucagon from the site of injection.

[0010] Thus, there is a need for a formulation of glucagon that provides a sustained release of glucagon into the circulation of a patient. Furthermore, there is a need for a formulation of glucagon more stable than currently marketed formulations, ideally one that retains activity for longer periods of time at room temperature. Such pharmaceutical preparations comprising glucagon would be useful for the preventive treatment of hypoglycemia and provide diabetic patients a means to reduce the risk of hypoglycemia while avoiding the complications of high glucose caused by excessive levels of glucagon. Ideally, the excipients used in these formulations would also be suitable for the preparation of other peptides and proteins in extended release formulations. The present invention meets these needs.

BRIEF SUMMARY OF THE INVENTION

[0011] The present invention relates to stabilized pharmaceutical preparations or formulations comprising a protein for which a sustained release profile is desired. In one embodiment, the protein is glucagon, and the formulation is suitable for parenteral administration and useful in the prevention of hypoglycemia. As used herein, a formulation has an "extended release" profile if, relative to another formulation, the time to reach the maximum concentration (Cmax) after administration is longer; or the time required for the concentration to decline to 50% of the Cmax after the Cmax has been achieved is longer.

[0012] In a first aspect, the invention provides a pharmaceutical preparation comprising a suspension of a protein and a stabilizing amount of a pharmaceutically acceptable cation. The protein in the suspension is in the form of a precipitate; the cation slows the dissolution of the precipitates. In one embodiment, the cation is present at a molar ratio in the range of 5 to 400 moles of cation per 1 mole of protein, hi one embodiment, the cation is present at a molar ratio in the range of 50 to 150 moles of cation per 1 mole of protein. Li one embodiment, the cation is present at a molar ratio of about 200 moles of cation per mole of protein, hi one embodiment, the cation is a multivalent cation, including but not limited to a divalent or trivalent cation, hi one embodiment, the cation is either a ferric, ferrous or calcium cation or a
combination of two or more of these cations. In one embodiment, the cation is a multivalent cation other than zinc. In one embodiment, the protein is glucagon. Glucagon is relatively insoluble at neutral pH but can be dissolved in aqueous media at either low pH (e.g., between pH 1-3) or high pH (e.g., between pH 9-11). The preparations of the invention allow for the sustained release of glucagon or other proteins into the body and ultimately the plasma of a patient injected with them. In one embodiment, the preparations retain activity of the active pharmaceutical ingredient (i.e., glucagon or another bioactive protein or peptide) at room temperature, e.g. 250C, for extended periods of time, such as a day, a week, or a month.

[0013] In one embodiment, the pharmaceutical preparations of the invention comprise, in addition to the suspension of protein and cation, a stabilizing amount of a pharmaceutically acceptable polymer. In one embodiment, the polymer is a polysaccharide. In one embodiment, the polysaccharide is sodium alginate. Alginic acid is a linear co-polymer of the two monomelic units α-L-guluronic acid and β-D- manuronic acid. The length of the polymer and the ratio of the two monomelic units can vary and variation in these properties alters the physico-chemical nature of the polymer. These preparations of the invention allow for the sustained release of a protein such as glucagon into the plasma of a patient injected with them. In one embodiment, the formulation retains glucagon activity at room temperature, e.g. 25°C, for an extended period of time, such as a day, a week, or a month. In one embodiment, the polymer is either of calcium alginate, CMC (sodium carboxymethyl cellulose), carbopol (containing polyacrylic acid), Gellan gum (containing polyglucuronic acid), or propylene glycol alginate. In one embodiment the polymer is a combination of two or more of these polymers.

[0014] In one embodiment, the pharmaceutical preparations of the invention comprise, in addition to the suspension of protein and cation and optional polymer, an anion such as sulfate, chloride, phosphate, tetraborate, citrate, tartrate, or alginate. The cation and anion may be added together (e.g., as a salt such as ferric chloride) or separately. Separate addition may be accomplished by, for illustration and not limitation, combining a first salt containing the cation (e.g., Fe2+) and a second salt containing the anion (e.g., Cl"), with the desired cation and desired anion being added at a ratio in the range of about 0.1 to about 10 moles cation per mole anion.
[0015] In one embodiment, the pharmaceutical preparation of the invention is in the form of a multi-use vial and comprises an additional excipient that inhibits microbiocidal activity. In one embodiment the excipient is selected from m-cresol, benzyl alcohol, methyl, ethyl, propyl and butyl parabens and phenol. In another embodiment, the pharmaceutical preparation of the invention is in the form of a single-use vial and optionally comprises an additional excipient that inhibits microbiocidal activity. In one embodiment the excipient is selected from m-cresol, benzyl alcohol, methyl, ethyl, propyl and butyl parabens and phenol.

[0016] In one embodiment, the pharmaceutical preparation of the invention is in lyophilized form and comprises an additional excipient that facilitates not only lyophilization but also rapid and complete resuspension during reconstitution of the preparation before use. In one embodiment, the resuspension-facilitating excipient is selected from disaccharides such as lactose, trehalose, and sucrose; sugar alcohols such as sorbitol or mannitol; polysaccharides such as the polymers commercialized as DEXTRAN such as DEXTRAN 40, 70 and 75, and FICOLL; and polyvalent alcohols, such as polyethylene glycol or polyvinyl alcohol; and combinations of two or more of these. In one embodiment, the resuspension-facilitating excipient is present in an amount of from 0.1 mg to 10 mg per mg glucagon. In one embodiment, resuspension-facilitating the excipient is lactose.

[0017] In one aspect, the present invention provides methods for making the pharmaceutical preparations of the invention. In one embodiment, the invention provides a method for the preparation of a pharmaceutical preparation comprising a protein and a stabilizing amount of a pharmaceutically acceptable cation, in which the protein is dissolved in water, in a solution of an excipient such as mannitol to provide for an isotonic solution, or in a solution of the cation, with or without excipient, at either high pH (e.g., pH greater than 9.0) or low pH (e.g., pH less than 3.0), optionally after sterile filtration. The solution is then returned to neutral pH, which leads to the formation of a stabilized suspension of the protein or peptide of interest, such as, for example, glucagon. The protein suspension can be lyophilized. hi one embodiment, the method provides a stabilized solution of glucagon. In one embodiment, the invention provides a method for the preparation of a pharmaceutical preparation comprising glucagon and a stabilizing amount of a pharmaceutically acceptable
cation, wherein glucagon is dissolved in a solution of the cation with or without excipient at either high or low pH optionally after sterile filtration. The solution is then returned to neutral pH. The suspension can then be lyophilized.

[0018] These and other aspects and embodiments of the invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 shows the pharmacokinetic (PK) profile in dogs of a solution of glucagon injected intravenously or subcutaneously.

[0020] Figure 2 shows the PK profile of glucagon, ferric sulfate glucagon, and ferric sulfate alginate glucagon suspension formulations injected into dogs. The figure demonstrates that glucagon precipitated in the presence of either ferric sulfate or ferric sulfate and sodium alginate has an extended PK profile relative to that of control glucagon and that glucagon precipitated in the presence of ferric sulfate plus alginate has a PK profile different from that of glucagon precipitated in the presence of ferric sulfate alone.

[0021] Figure 3 shows the PK profile of glucagon ferric sulfate and alginates with different physico-chemical properties. Alginic acid is a linear co-polymer of the two monomelic units α-L-guluronic acid and β-D-manuronic acid. The length of the polymer and the ratio of the two monomelic units can vary and variation in these properties alters the physico-chemical nature of the polymer. LVG alginate is a low viscosity (relatively shorter) polymer with a higher concentration of α-L-guluronic acid. MVG is a medium viscosity (relatively longer) polymer with a higher concentration of α-L-guluronic acid. LVM alginate is a low viscosity (relatively shorter) polymer with a higher concentration of β-D-manuronic acid. MVM is a medium viscosity (relatively longer) polymer with a higher concentration of β-D- manuronic acid. Glucagon ferric sulfate LVG alginate (1 mg/ml), glucagon ferric sulfate MVG alginate (1 mg/ml), glucagon ferric sulfate LVM alginate (1 mg/ml), and glucagon ferric sulfate MVM alginate (1 mg/ml) formulations were prepared and injected into dogs. The figure demonstrates that the PK profile of glucagon precipitated in the presence of 40 inM ferric sulfate changes with the form of the sodium alginate added to the formulation.
[0022] Figure 4 shows the PK profile of glucagon zinc chloride (4OmM) LVG alginate (1 mg/ml), glucagon calcium chloride LVG alginate (1 mg/ml) and glucagon LVG alginate (1 mg/ml) formulations injected into dogs. The figure demonstrates that the PK profile of glucagon precipitated in the presence of calcium is different from that of glucagon precipitated in the presence of zinc and that both are different from glucagon precipitated in absence of added cation.

[0024] The present invention provides a pharmaceutical preparation comprising a protein and a stabilizing amount of a pharmaceutically acceptable cation. In one embodiment, the cation is a multivalent cation, including but not limited to divalent and trivalent cations. In one embodiment, the cation is selected from the group consisting of ferric, ferrous and calcium cations and combinations of two or more of these cations. In one embodiment, the cation is a multivalent cation other than zinc, hi one embodiment, the invention further comprises one or more additional excipients. These preparations allow for the sustained release of a protein or peptide into the plasma of a patient injected with the preparation. Furthermore the preparations retain activity at room temperatures, e.g. 250C, for extended periods of time. In one embodiment, the pharmaceutical preparation is a pharmaceutical formulation of glucagon in suspension form, hi one embodiment the suspension is a colloidal suspension. Colloidal suspensions may be made by art-known methods. High speed homogenization or a microfluidizer may be used to yield small particle sizes, hi another embodiment, the pharmaceutical preparation is a pharmaceutical formulation of glucagon in suspension form that has been lyophilized.

[0025] A cation in accordance with the present invention is a pharmaceutically acceptable cation, hi one embodiment, the cation is multivalent, hi various
embodiments, the cation is a ferric, ferrous or calcium cation or a mixture of two or more of these. In one embodiment, the pharmaceutical preparation of the invention comprises ferric, ferrous or calcium cation in an amount of about 1 to 100, often 2 to 100 micromoles per mg glucagon, e.g., about 50 micromoles per mg glucagon. In one embodiment, the pharmaceutical preparation of the invention comprises cation in an amount of about 10 to 40 micromoles per mg glucagon. The amount of ferric, ferrous or calcium cation is in some embodiments about 40 micromoles per dose, where the glucagon dose is about 0.4 mg. The amount of ferric, ferrous or calcium cation is in some embodiments about 6 to 25, often 7 to 15 micromoles per dose, where the glucagon dose is about 0.150 mg. hi another embodiment, the cation is selected from the group consisting of magnesium, manganese, nickel, copper, and selenium. In one embodiment, the cation is in the form of a salt and complexed with an anion selected from the group consisting of sulfate, chloride, acetate phosphate, tetraborate, citrate, tartrate, and alginate anions, hi one embodiment, the salt is ferric sulfate, hi another embodiment, one or more of these anions is added to the preparation separately from the cation. In one embodiment, the anion is a sulfate, chloride or acetate anion, and the protein is glucagon.

[0026] A pharmaceutical preparation of the invention can also comprise a pharmaceutically acceptable polymer, e.g. for decreasing the rate of dissolution of the glucagon suspension after the suspension has been administered to a patient, hi various embodiments, the polymer is a polysaccharide that is a salt or acid form of alginate in an amount in the range 0.1 to 2.5 mg per mg protein, sometimes in the range 0.4 to 1.5 mg, usually in the range 0.5-1 mg. In one embodiment, about 1 mg alginate per mg of a protein such as glucagon is used. The amount of alginate is in some embodiments about 0.5 mg per dose, hi another embodiment, the polymer is selected from the group consisting of, CMC (sodium carboxymethyl cellulose), carbopol (containing polyacrylic acid), Gellan gum (containing polyglucuronic acid), and propylene glycol alginate

[0027] A pharmaceutical preparation of the invention in lyophilized form can also comprise one or more protamines or other small negatively charged proteins, e.g. for either increasing or decreasing the rate of dissolution of the glucagon suspension after the suspension has been administered to a patient, hi one embodiment, a protamine is
present in an amount of from 0.5 to 2 mg per mg glucagon, often about 1 mg per mg glucagon.

[0028] A pharmaceutical preparation of the invention in lyophilized form can also comprise one or more additional excipients, e.g. for facilitating the lyophilization and/or the rapid and complete resuspension thereof when reconstituting the preparation before use. In various embodiments, an excipient useful in accordance with the present invention is selected from pharmaceutically acceptable disaccharides, including but not limited to lactose, trehalose, and sucrose; sugar alcohols, including but not limited to sorbitol or mannitol; polysaccharides such as the polymers commercialized as DEXTRAN products, including but not limited to Dextran 40, 70 and 75, and FICOLL; and polyvalent alcohols, including but not limited to polyethylene glycol, polyvinyl alcohol, and combination of two or more of these. In one embodiment, the excipient is a mixture of one or more disaccharides, sugar alcohols, polysaccharides, DEXTRAN, and polyvalent alcohols. The excipient is in some embodiments present in an amount of from 10 to 600 micromoles per mg glucagon. In one embodiment, the excipient is lactose.

[0029] In one embodiment, the pharmaceutical preparation of the invention is a stabilized suspension of glucagon. In various embodiments, the pH of the preparations of the invention that are in the form of a solution is adjusted to a pH in the pH interval 4-9. hi some embodiments, the pH is adjusted to a pH in the interval 5-9, alternatively 5-8, and in one embodiment, the pH is adjusted to about 6.8.

[0030] The invention also provides a method for the preparation of a pharmaceutical preparation comprising glucagon and a stabilizing amount of a pharmaceutically acceptable cation, wherein the glucagon suspension is prepared in the presence of the cation. In one embodiment, an excipient is also present in the solution. In one embodiment, the method further comprises a lyophilization step. In one embodiment, the solution is lyophilized after sterile filtration.

[0031] In one embodiment, the pharmaceutical preparation according to the invention comprises glucagon; sodium alginate; and ferric, ferrous or calcium or a mixture of two or more of these as a stabilizing agent. Sodium alginate consists mainly of the sodium salt of alginic acid, which is a mixture of polyuronic acids
composed of 1, 4'-linked β-D-mannuronic acid and β-L-guluronic acid residues. The polyvalent cations can replace the monovalent cation sodium and in so doing form more complex and ordered structures (such as gels and sheets) than those formed in the presence of monovalent cations. These structures can entrap the glucagon suspension, reducing the rate of dissolution of the glucagon suspension, hi this manner, the invention provides a formulation that has an extended release profile and is stable at room temperature.

[0032] hi one embodiment, the invention provides a unit dose form of glucagon suitable for subcutaneous injection that is intended to be administered to a diabetic patient on insulin so that hypoglycemia can be prevented for an extended period of time (6 to 12 hours), hi one embodiment, such a unit dose is administered in the evening to provide protection from hypoglycemia during the night, hi one embodiment, the unit dose form is a vial containing 0.15 to 0.25 milligrams of glucagon, 0.2 to 0.5 mg ferric salt (e.g., ferric sulfate), and 0.15 milligrams of sodium alginate, hi one embodiment, the unit dose form is a vial containing about 0.150 milligrams of glucagon., 0.2 to 0.5 mg ferric salt (e.g., ferric sulfate), and 0.15 milligrams of sodium alginate. In one embodiment, the unit dose form is a vial containing 0.4 milligrams of glucagon, 1.2 milligrams of ferric chloride, and 0.4 milligrams of sodium alginate. If lyophilized, the drug product in the vial may be reconstituted, e.g., by the addition of 0.2 milliliters of 5% mannitol to a volume of 0.2 milliliters, prior to administration, hi one embodiment, the vial can contain the formulation of the drug in solution; such vials may be stored under conditions that enhance stability, i.e., at 4°C, until use.

[0033] hi one embodiment, the invention provides a multi-dose formulation of glucagon or other protein or peptide suitable for subcutaneous injection. In one embodiment, this multi-dose formulation provides glucagon intended for administration to a diabetic patient on insulin, for example and without limitation, in the evening so that hypoglycemia can be prevented during the night (6 to 10 hours), hi one embodiment, the multi-dose formulation is in a vial containing 4 milligrams of glucagon, 12 milligrams grams of ferric chloride, 4 milligrams of sodium alginate. In one embodiment, the multi-dose form is a vial containing 4 milligrams of glucagon, 12 milligrams grams of ferric chloride, 4 milligrams of sodium alginate, and a suitable
preservative (e.g., m-cresol). The drug product in the vial can be in lyophilized form, and if so, then can be reconstituted, for example, by the addition of 2 milliliters of 5% mannitol to a volume of 2 milliliters prior to administration. This multi-use vial provides up to ten injections of approximately 0.2 milliliters per injection. In a related embodiment, the multi-dose formulation is in a vial containing 1.5 milligrams of glucagon, 2-5 milligrams grams of ferric sulfate, and 1.5 milligrams of sodium alginate and optionally other excipients. This multi-use vial provides for up to ten injections each of approximately 0.2 milliliters per injection.

[0034] The amino acid sequence of human glucagon is identical to the amino acid sequence of porcine and bovine glucagon. Hence, glucagon may be isolated by conventional extraction form porcine or bovine pancreatic glands. In the alternative, glucagon may be prepared by fully or partially chemical synthesis or by recombinant techniques, e.g. as disclosed in U.S. Pat. No. 4,826,763. Glucagon can also be purchased from a number of pharmaceutical agent manufacturers. Those of skill in the art recognize that the present invention also provides stabilized compositions of glucagon derivatives and muteins. Other proteins that may be formulated as described herein include GLP-I, xenatide, human growth hormone, insulin, interferons, and erythropoetin. Particular therapeutic proteins that may be formulated according to the present method include proteins that are relatively insoluble at neutral pH. Formulations of other proteins may be accomplished according to the invention provided the pH at which they precipitate is in the range of acceptable pH for injections (i.e., in the range of pH 3 to slightly above neutral pH). In one embodiment, the therapeutic protein is approved for subcutaneous or intravenous administration.

[0035] Those of skill in the art will recognize that the pharmaceutical preparations of the invention may be administered in any way appropriate for the patient and condition to be treated. In one embodiment, the mode of administration is parenteral. In one embodiment, the mode of administration is subcutaneous or intramuscular injection. In another embodiment, the formulation is administered intravenously. Other modes of administration include administration to a mucosal membrane, e.g. nasal administration. The pharmaceutical preparations of the invention include those in the form of a unit dose contained in a vial or cartridge or other suitable container.
[0036] The formulations of the invention find particular use in treating a patient with diabetes according to the methods described in U.S. Pat. No. 7,314,859 and U.S. Pat. App. Pub. No. 20060014670. As described therein, administration of very low doses of glucagon as part of a treatment regimen significantly reduces the likelihood the diabetic patient will suffer from nocturnal hypoglycemia. In one embodiment, a very low dose of glucagon (e.g., 150 micrograms) formulated as described herein is administered at bedtime.

[0037] The invention is described by way of example in the Examples below, which illustrate but are not to be considered as limiting the scope of the invention defined by the appended claims that follow them.

EXAMPLES

[0038] These examples illustrate a number of different glucagon formulations of the invention and describe the PK profile of these formulations as measured in rats and dogs, together with the PK profiles of a control formulation of glucagon that has not been formulated for extended release.

EXAMPLE 1 Preparation of Glucagon Formulations

[0039] Glucagon can be purchased as a synthetic 29 amino acid peptide from commercial suppliers such as American Peptide (777 East Evelyn Ave, Sunnyvale, CA 94086 USA). Glucagon solutions may be prepared by a variety of methods, including the following methods, hi the method used to prepare the glucagon control solution referenced in the figures herein, glucagon is dissolved in isotonic mannitol (about 5%) at low pH (e.g., pH 1-3, often about pH 2.5) to provide the desired concentration. The pH can be adjusted with hydrochloric acid, acetic acid or other acid. The solution is filtered through a 0.2 um filter into a sterile container. A glucagon solution can also be prepared at high pH in which glucagon is dissolved in isotonic mannitol (about 5%) at high pH (e.g. pH 9-11, often about 10.0) to provide the desired concentration. The pH can be adjusted with NaOH or other base.

[0040] The glucagon suspension can be formed from the low pH solution by adjusting the pH to neutral (range of 6.5 to 7.5) by addition of sodium hydroxide. A
fixed sodium hydroxide solution (2-8 M) is filtered through a 0.2 micron filter and added into the low pH glucagon solution with constant mixing so that the final pH is in the range 6.5 to 7.5.

[0041] In another method, the suspension of glucagon can be prepared at a concentration of approximately 10 mg/ml from a solution of glucagon that is prepared in water containing 5% mannitol that has been adjusted to high pH (e.g., pH 10) with sodium hydroxide. A volume of 1 M HCl is added to achieve a final pH of about 6.5 to 7.5. The glucagon can then be filled into glass vials with constant mixing.

[0042] Suspensions of glucagon containing cations and alginate can be prepared as follows. Prepare a solution of glucagon at approximately 10 mg/ml in water that has been adjusted to a pH of 10 with sodium hydroxide. Prepare a solution of 10% (weight/volume) mannitol in water. Prepare a IM ferric sulfate (or 1 M ferric chloride or 1 M calcium chloride or 1 M zinc acetate) solution. Prepare a 5M NaOH solution. Prepare a solution of 10 mg/ml sodium alginate in water. Filter all solutions into sterile glass containers. Mix the stock solutions (glucagon, mannitol, ferric sulfate, sodium hydroxide, sodium alginate (sodium alginate can be purchased from commercial suppliers such as Sigma-Aldrich Corp (St. Louis, MO)) in the following order with constant stirring to prepare a suspension with the components at the indicated concentration and pH: (1) Glucagon at pH 10 so that the final concentration is 1 mg/ml; (2) Mannitol solution so that the final concentration is 5%; (3) Sodium alginate solution (if sodium alginate is present) so that the final concentration is 1 mg/ml; (4) ferric sulfate (or ferric chloride or calcium chloride or zinc acetate) so that the final concentration is 40 mM (or 20 inM or 10 mM); and (5) 5 N HCl to achieve a final pH of about 6.5 to 7.5. The glucagon formulation can then be filled into glass vials with constant mixing.

EXAMPLE 2 Pharmacokinetic Profiles of GlucaRon Formulations in Dogs [0043] The pharmacokinetic profile of a control glucagon solution or the glucagon suspensions prepared as described in Example 1 can be determined in dogs by the following procedures. One group of 4 male beagle dogs is used for the study. On Day 0, the animals are weighed. Dosages are calculated based on body weight and the animals are dosed subcutaneously (sc) or intravenously (iv) with either the glucagon
control solution or a glucagon suspension. Blood is collected from each dog prior to dosing ("0 min") and at 5, 10, 15, 30, and 60 min, and 2, 4, 6, 8, 10 and 24 hrs following the dose administration (for a total of 12 time points). At each time point, blood (0.3 mL/sample) is collected and placed into labeled Microtainer® tubes with sodium heparin as the anti-coagulant. The heparinized blood is centrifuged, and the supernatant pipetted into another set of labeled Eppendorf® tubes (for at least 0.1 mL plasma) and frozen at or below -70 0C. Glucagon in the plasma samples can be measured using a radioimmune assay (available from Millipore, St. Louis MO).

[0044] The pharmacokinetic profiles of the pharmaceutical preparations of glucagon prepared as described in Example 1 was determined in rats and dogs. The results of the dog studies are shown in Figures 1-5. Collectively, the figures show that a suspension of glucagon has an extended release profile as compared to a solution of glucagon and that the addition of a cation provides a formulation of glucagon in suspension that has an extended release profile as compared to a suspension of glucagon prepared in the absence of added cation.

[0045] In rats {see provisional application No. 60/900,509 incorporated herein by reference), the use of the ferric or calcium cation provides an extended release profile relative to the control formulation. Use of sulfate as the anion (as compared to chloride) can produce a preparation with a PK profile in rats that has a lower maximum concentration but a higher concentration at later time points. It was also observed that the use of calcium as the cation (as compared to ferric) can produce a preparation that has a release profile that is intermediate between the control suspension prepared in the absence of exogenous cations and the suspension formed in the presence of ferric cations. The presence of alginate can provide a preparation that has a release profile that is further extended, relative to control formulations and certain other formulations, in rats and dogs.

EXAMPLE 3 PK Profiles of Different Glucagon Formulations in Dogs [0046] As noted above, the pharmacokinetic profiles in dogs of the different pharmaceutical preparations of glucagon prepared as described in Example 1 are shown in Figures 1-5.
[0047] Figure 1 shows the pharmacokinetic profile of glucagon in dogs following either the intravenous or subcutaneous injection of a glucagon solution. These pharmacokinetic profiles provide the baseline from which the extended release PK of the cation-glucagon suspensions are demonstrated. In Figure 1, the glucagon solutions are made as described in Example 1 (low pH method, without cation or alginate). A glucagon solution in 5% mannitol (pH adjusted to pH 2.5 with 5 N HCl) was made by adding 20 mg of the glucagon powder to sufficient 5% mannitol (adjusted to pH 2.5 with IM HCl) so as to make a 5 mg/ml solution (4 ml total volume).

[0048] Each dog was weighed and injected with 0.25 ml of the 5 mg/ml solution (1.25 mg/dog, approximately 0.125 mg/kg). For the dogs injected intravenously, blood was collected pre-dose and at 2, 5, 10, 15, 20, 30, 40, 60 minutes post-dose and at 2, 3 and 4 hours post-dose. For the dogs injected subcutaneously, blood was collected pre-dose and at 5, 10, 15, 30, 60 minutes post-dose and at 2, 4, 6, 8, 10 and 24 hours post-dose. Glucagon concentration in the plasma was determined as described in Example 2, and the pharmacokinetic profile shown in Figure 1 was obtained from analysis of the results.

[0049] Figure 2 shows results of a study in which the pharmacokinetic profiles in dogs administered one of three different glucagon suspensions were compared. In brief, these three suspensions were made with either no exogenous cation added (the control suspension), with 4OmM ferric sulfate added, or with 40 mM ferric sulfate plus 1 mg/ml sodium alginate added. The control suspension was prepared as follows: three hundred and sixty microliters of acetic acid were added to 24 ml of water, and 1.5 g mannitol were added and mixed to dissolve. Glucagon (186.8 mg) was added and mixed to dissolve. The total volume was adjusted to 30 ml with water. The measured pH was 2.61. This solution was filtered through a 0.22 micron filter into a sterile, glass vial. 1.23 niL of 5 N NaOH solution was added to the glucagon solution with agitation. The measured pH was 6.3. The glucagon suspension containing 40 mM ferric sulfate was prepared by adding 360 microliters of acetic acid, 186.8 milligrams of glucagon and 1.5 g of mannitol to 24 ml of water and mixing to dissolve. 1 ml of a IM ferric sulfate solution was added. 1.67 ml of 5M NaOH were added. The total volume was adjusted to 30 ml with water. The measured pH was 6.5. The suspension containing 40 mM ferric sulfate and 1 mg/ml of sodium alginate was
prepared adding 0.65 ml of 5N NaOH to 14 ml of water and adding 123.6 mg of glucagon and mixing to dissolve. Two ml of a sodium alginate (10 mg/ml) suspension were added and mixed. This suspension was filtered through a 0.22 micron filter. ImI ferric sulfate (IM) solution was added. 0.75 ml of 5M HCl was added and mixed. The measured pH was 2.67. Then, 0.6 ml 5M NaOH was added. Then, 0.4 ml 5M HCl was added. Water was added to achieve a final volume of 25 ml. The final pH was 6.67. Sodium alginate was purchased from Sigma Aldrich.

[0050] These three suspensions were injected subcutaneously into Beagle dogs at a dose of 0.25 mg/kg. Blood samples were taken pre-dose and at 5, 10, 15, 30 and 60 minutes and at 2, 4, 6 and 24 hours post-dose. The glucagon present in the plasma was determined by Millipore Inc. using an RIA. As can be seen in comparing the PK profiles of these three suspensions (Fig. 2) to the PK profile of the control glucagon solutions shown in Fig. 1 , the half life of the glucagon in the plasma is extended for the suspensions. The half life was extended by the presence of 40 mM ferric sulfate and further by the additional presence of 1 mg/ml sodium alginate.

[0051] Figure 3 shows the PK profile of glucagon suspensions prepared in the presence of 40 mM ferric sulfate using different forms of sodium alginate. The four forms are: low viscosity high-mannuronic acid (LVM), medium viscosity high- mannuronic acid (MVM), low viscosity high-guluronic acid (LVG), medium viscosity high-guluronic acid (MVG). These four forms of alginate can be purchased from FMC BioPolymer AS (Drammen, Norway). 10 mg/ml suspensions of these alginates were prepared. A high pH glucagon solution was prepared as described in Example 1 , and a volume of one of the four alginate forms (prepared as a suspension at 10 mg/ml in water) sufficient to achieve a final concentration of 1 mg/ml added. To this is added a volume of 1 M ferric sulfate sufficient to achieve a final concentration of 40 mM. To this is added a volume of 1 M HCl to achieve a neutral pH (6.0 to 7.5). These four suspensions were injected subcutaneously into Beagle dogs at a dose of 0.05 mg/kg, and blood samples were taken pre-dose and at 5, 10, 15, 30 and 60 minutes and at 2, 4, 6, 8, 10 and 24 hours post-dose. The glucagon present in the plasma was determined by Millipore Inc. using an RIA. As can be seen by comparing the PK profiles of these four suspensions to the PK profile of the glucagon solution shown in Fig. 1, the half life of the glucagon from the four suspensions in the plasma
is extended, and the PK profile can be manipulated by altering the viscosity and the mannuronic acid content.

[0052] Figure 4 shows the PK profile of glucagon suspensions prepared in the presence of 1 mg/ml LVG alginate and either no added cation, 40 mM zinc acetate or 40 mM calcium chloride. A glucagon solution was prepared at high pH as described in Example 1, and a volume of LVG alginate (prepared as a 10mg/ml suspension in water) sufficient to achieve a final concentration of 1 mg/ml was added. To this is added a volume of 1 M calcium chloride or IM zinc acetate sufficient to achieve a final concentration of 40 mM. The control suspension had no added cation solution. To these solutions is added a volume of 1 M HCl to achieve a neutral pH (6.0 to 7.5). These three suspensions were injected subcutaneously into Beagle dogs at a dose of 0.05 mg/kg and blood samples taken pre-dose and at 5, 10, 15, 30 and 60 minutes and at 2, 4, 6, 8, 10 and 24 hours post-dose. The glucagon present in the plasma was determined by Millipore Inc. using an RIA. As can be seen in comparing the PK profiles of these three suspensions to the PK profile of the glucagon solution shown in Fig. 1, the half life of the glucagon from the suspensions in the plasma was extended and could be altered by changing the cation present in the suspension.

[0053] Figure 5 shows the results of a study in which glucagon was prepared as a precipitate in the presence of either 40 mM, 20 mM, 1OmM or zero ferric sulfate and sodium alginate (LVG form) at 1 mg/ml. The data described in this figure for 0 mM ferric sulfate and 1 mg/ml sodium alginate is the same data as that in Fig. 4 for 1 mg/ml sodium alginate (no added cation). A glucagon solution was prepared at high pH as described in Example 1, and a volume of LVG alginate (prepared as a 10mg/ml suspension in water) sufficient to achieve a final concentration of 1 mg/ml was added. To this is added a volume of 1 M ferric sulfate sufficient to achieve a final concentration of 40 mM or 20 mM or 1OmM ferric sulfate. The control suspension had no added ferric sulfate solution. To these solutions is added a volume of 1 M HCl to achieve a neutral pH (6.0 to 7.5). These four suspensions were injected subcutaneously into Beagle dogs at a dose of 0.05 mg/kg and blood samples taken pre-dose and at 5, 10, 15, 30 and 60 minutes and at 2, 4, 6, 8, 10 and 24 hours post- dose. The glucagon present in the plasma was determined by Millipore Inc. using an RIA. As can be seen in comparing the PK profiles of these three suspensions to the
PK profile of the glucagon solution shown in Fig. 1, the half life of the glucagon from the suspensions in the plasma is extended and can be altered by changing the concentration of ferric sulfate present in the suspension.

EXAMPLE 5 Prevention of Hypoglycemia Using Very Low Dose Glucagon [0054] This example describes treating a patient with diabetes to prevent insulin- induced hypoglycemia by administering a glucagon formulation of the invention according to the methods described in US Pat. No. 7,314,859 and U.S. Pat. App. Pub. No. 20060014670. As described therein, administration of very low doses of glucagon as part of a treatment regimen significantly reduces the likelihood the diabetic patient will suffer from nocturnal hypoglycemia.

[0055] The patient receives her normal daily dose(s) of insulin, which can be administered by any route (e.g., subcutaneously) and which may be a long-acting form of insulin. At bedtime, a glucagon suspension formulation of the invention (e.g., 0.15 mg glucagon, 30 mM ferric sulfate, and 1 mg/ml sodium alginate in 0.2 ml sterile buffered solution) is administered subcutaneously by injection or via pump. The patient's blood sugar levels are maintained at euglycemic levels for at least 6 hours.

Claims

CLAIMS:

1. A pharmaceutical formulation comprising a protein and a stabilizing amount of a pharmaceutically acceptable cation, wherein said cation is present at a molar ratio in the range of 5 to 400 moles cation: 1 mole protein.

2. The pharmaceutical formulation of Claim 1 wherein the cation is multivalent.

3. The pharmaceutical formulation of Claim 2 wherein the protein is glucagon.

4. The pharmaceutical formulation of Claim 3, wherein the cation is selected from the group consisting of ferric, ferrous and calcium cations.

5. The pharmaceutical formulation of Claim 3, wherein the cation is selected from the group consisting of magnesium, manganese, nickel, copper and selenium.